[0001] The present invention relates to the enantiomeric enrichment and stereoselective
synthesis of chiral amines.
Background of the Invention
[0002] The biological activity of chemical products such as pharmaceuticals and agricultural
products which possess a center of chirality often is found to reside principally
in one of the possible chiral forms. Since most chemical syntheses are not inherently
stereoselective, this poses a serious chemical processing problem. Enrichment in favor
of one chiral form thus will be required at some stage, either the final chiral compounds
or chemical precursors which possess the same center of chirality. Whatever stage
is selected for the enrichment, and in the absence of a method of recycling of the
unwanted enantiomer, the process is inherently limited to a maximum theoretical yield
of 50% for the desired enantiomer.
[0003] Many of the chiral compounds of this type are amines. Moreover because of their synthetic
versatility, amines also are good candidates for resolution, after which stereoselective
conversion to the chiral compound can be effected. Chemical production of a chiral
amine free of its enantiomer heretofore has relied largely on resolution of a mixture
of the two chiral forms through formation of diastereomeric derivatives such as a
salt with a chiral acid, stereoselective syntheses, or the use of chiral chromatographic
columns. See for examples U.S. Patent No. 3,944,608 and EP-A 36,265.
[0004] Some structural types of amines lend themselves to enzymatic resolution. Enzymatic
reactions involving α-amino acids are well known and their use has been proposed for
stereospecific preparations. U.S. Patent No. 3,871,958, for example, discloses the
enzymatic preparation of derivatives of the α-amino acid serine by coupling an aldehyde
with glycine in the presence of a threoninealdolase, derived from an
E. coli species, as well as a related synthesis of serinol employing ethanolamine.
[0005] Relatively little has been reported on enzymatic reactions on amino acids in which
the amino group is not vicinal to a carboxylic acid group. Yonaha et al.,
Agric. Biol. Chem.,
42 (12), 2363-2367 (1978) describe an omega-amino acid:pyruvate transaminase found in
a
Pseudomonas species for which pyruvate was the exclusive amino acceptor. This enzyme, which had
been previously crystallized and characterized {see Yonaha et al.,
Agric. Biol. Chem.,
41 (9), 1701 1706 (1977)} had low substrate specificity for omega amino acids such as
hypotaurine, 3 aminopropane sulfonate, β-alanine, 4-aminobutyrate, and 8-aminooctanoate
and catalyzed transaminations between primary aminoalkanes and pyruvate.
[0006] Nakano et al.,
J. Biochem.,
81, 1375-1381 (1977) identified two omega-amino acid transaminases in
B. cereus: a β-alanine transaminase, which corresponds to Yonaha et al.'s omega-amino acid:pyruvate
transaminase, and a -aminobutyrate transaminase. The two could be distinguished by
their dramatically different activities on β-alanine (100 vs. 3) and -aminobutyrate
(43 vs.100), respectively, as well as their distinct amino acceptor requirements.
[0007] Burnett et al.,
J.C.S. Chem. Comm., 1979, 826-828, suggested omega-amino acid:pyruvate transaminase and -amino butyrate
transaminase exhibit different preferences for the two terminal hydrogen atoms in
tritium labelled -aminobutyrate.
[0008] Tanizawa et al.,
Biochem. 21, 1104-1108 (1982) examined bacterial L-lysine-ε-aminotransferase and L-ornithine-δ-aminotransferase
and noted that while both are specific for L-amino acids, they act distally and with
the same stereospecificity as the -aminobutyrate transaminase studied by Burnett
et al.,
supra.
[0009] Yonaha et al.,
Agric. Biol. Chem.,
47 (10), 2257-2265 (1983) subsequently characterized omega-amino acid:pyruvate transaminase
and -aminobutyrate transaminase (EC 2.6.1.18 and EC 2.6.1.19) and documented their
distribution in a variety of organisms.
[0010] Waters et al.,
FEMS Micro. Lett., 34 (1986) 279-282, reporting on the complete catabolism of β-alanine and β-aminoisobutyrate
by
P. aeruginosa, noted that the first step involved transamination with β-alanine:pyruvate aminotransferase.
[0011] Enzymatic methods have been considered as a method for separating mixtures of chiral
amines which are not amino acids, as for example 2-aminobutanol. Most of these involve
derivatization, particularly of the amino group, and utilization of this protected
group or another group in the molecule to effect separation. EP-A 222,561, for example,
describes a process in which racemic 2-aminobutanol is converted to an N-carbamoyl
derivative which then is brought into contact with an alkyl alkanoate in the presence
of a lipase enzyme. Esterification of the free hydroxy group apparently is limited
to the S-enantiomer of the N-carbamoyl derivative, which is thereafter hydrolysed.
This process of course is necessarily limited to amines carrying an esterifiable
hydroxy group and, moreover, specifically requires prior protection of the amino group
through formation of -NH-CO- carbamoyl group in order to obtain stereospecificity
in enzymatic reaction.
[0012] EP-A 239,122 describes a similar process applicable to the broader class of 2-amino-1-alkanols.
[0013] Japanese Kokai JP 55-138,389 describes the preparation of vicinal amino alcohols
by subjecting an alkyl or aralkyl substituted ethyleneimine to microorganisms of the
genus
Bacillus,
Proteus,
Erwinia, or Klebsiella.
[0014] Japanese Kokai JP 58-198,296 discloses a process in which
d,
l N-acyl-2-aminobutanol is subjected to the action of an aminoacylase derived from
various species of
Asperigillus,
Penicillium, and
Streptomyces which hydrolyses only the
d-N-acyl-2-aminobutanol.
[0015] Japanese Kokai JP 59-39,294 describes a process for resolving racemic 2-aminobutanol
through formation of an N-acetyl derivative which is treated with a
Micrococcus acylase to give
l-2-aminobutanol and
d-N-acetyl-2-aminobutanol, the latter then being chemically hydrolysed to afford
d-2-aminobutanol.
[0016] Japanese Kokai JP 63-237796 describes a process in which R,S-1 methyl-3-phenylpropylamine
is cultured aerobically in a variety of specified microorganisms with the S-form
being metabolized preferentially. The highest yields and optical purity is reported
for the yeast species
Candida humicola and
Trichosporon melibiosaceum. The enzymatic nature of the metabolism of the S-form which occurs in these aerobic
cultures,
e.
g., an oxidase, dehydrogenase, ammonia lysase, etc., is not indicated.
[0017] The abstract of Japanese Kokai JP 63-273486 discloses the microbial synthesis of
1-(4-methoxyphenyl)-2-aminopropane with the R-configuration at one of the two chiral
centers from 1-(4-methoxyphenyl)-2-propanone with
Sarcina lutea.
Detailed Description
[0018] In its broadest sense, the present invention involves the use of an omega-amino acid
transaminase in the presence of an amino acceptor to enantiomerically enrich a mixture
of, or to stereoselectively synthesize, chiral amines in which the amino group is
bound to a non-terminal, chirally substituted, carbon atom. Thus the invention is
based on the discovery that omega-amino acid transaminases operate stereoselectively
on amino groups which are not in an omega position and that this action can be used
both for enantiomeric enrichment of a mixture of chiral amines and stereoselective
synthesis of a chiral amine of only one configuration.
[0019] By the term omega-amino acid transaminases is meant any enzyme which exhibits the
property of converting the terminal -CH₂-NH₂ group of an omega-amino acid to a -CH=O
group.
[0020] The enzymatic equilibrium reaction involved in the present invention can be depicted
as follows:

in which each of R¹ and R², when taken independently, is an alkyl or aryl group which
is unsubstituted or substituted with one or more enzymatically non-inhibiting groups
and R¹ is different from R² in structure or chirality, or R¹ and R², taken together,
are a hydrocarbon chain of 4 or more carbon atoms containing a center of chirality.
[0021] As used herein, "amino acceptor" refers to various carbonyl compounds, more fully
discussed below, which are capable of accepting an amino group from the depicted
amine under the influence of an omega-amino acid transaminase. "Amino donor" refers
to various amino compounds, more fully discussed below, which are capable of donating
an amino group to the depicted ketone, thereby becoming a carbonyl species, also under
the influence of the same omega-amino acid transaminase.
[0022] The enzymatic reaction depicted above is characterized firstly by the fact that the
omega-amino acid transaminase operates on a primary amine in which the amino group
is not in an omega (or terminal) position. Secondly, the transaminase operates on
an amine which need not be an amino acid. Thirdly, the consumed amine product of the
enzymatic transformation is not irreversibly metabolized but can be stereoselectively
reconverted to the starting amine of a uniform chirality.
[0023] In a first embodiment, the present invention provides a process for the enantiomeric
enrichment of a mixture of chiral amines of the formula:

in which each of R¹ and R² are as defined above through the action of an omega-amino
acid transaminase in the presence of an amino acceptor. As can be seen, the compounds
of Formulas IA and IB are enantiomers (or diastereomers if either R¹ or R² contains
a second chiral center) and are chiral by reason of R¹ being different in structure
or chirality from R².
[0024] In a second embodiment, the invention provides a process for the stereoselective
synthesis of one chiral form of an amine of formula IA or IB in an amount substantially
greater than the other by subjecting a ketone of the formula:
R¹-

-R² II
in which R¹ and R² are as defined above to the action of an omega-amino acid transaminase
in the presence of an amino donor.
[0025] Both embodiments are based on the discovery that the action of an omega-amino acid
transaminase is not limited to omega-amino groups and moreover is largely or exclusively
stereoselective with respect to amines of the defined class, converting only one chiral
form of the amine to the corresponding ketone which is no longer chiral (at least
with respect to the carbonyl carbon atom) and in turn converting that ketone to only
one chiral form of the amine.
[0026] The term "enantiomeric enrichment" as used herein refers to the increase in the amount
of one enantiomer as compared to the other. This can involve (i) a decrease in the
amount of one chiral form as compared with the other, (ii) an increase in the amount
of one chiral form as compared with the other, or (iii) a decrease in the amount
of one chiral form and an increase in the amount of the other chiral form. A convenient
method of expressing the enantiomeric enrichment achieved is the concept of enantiomer
excess, or "ee", expressed by the expression:

in which E¹ is the amount of the first chiral form of the amine and E² is the amount
of the second chiral form of the same amine. Thus if the initial ratio of the two
chiral forms is 50:50 and an enantiomeric enrichment sufficient to produce a final
ratio of 50:30 is achieved, the ee with respect to the first chiral form is 25%,
whereas if the final ratio is 70:30, the ee with respect to the first chiral form
is 40%. Typically with the process of the present invention, ee's of 90% or greater
can be achieved.
[0027] "Substantially greater" as used herein with reference to the stereoselective synthesis
of one chiral form of an amine over the other refers to a ratio of at least about
3:1, representing an ee of at least about 50%.
[0028] The chiral amines of Formulas IA and IB employed in the present process have several
structural restraints. First while the amino group is a primary amine, it must be
bound to a secondary carbon atom;
i.
e., a carbon atom carrying one hydrogen atom and two substituents which are other than
hydrogen (R¹ and R²). Secondly, while R¹ and R² are selected from the same types of
structure, these groups must render the molecule chiral;
i.
e., R¹ necessarily will be different from R² in structure or chirality or R¹ and R²
when taken together are a chiral group. Generally when taken independently, R¹ and
R² will be alkyl, aralkyl, or aryl groups, preferably a straight or branched alkyl
group of from 1 to 6 carbon atoms, a straight or branched phenylalkyl group of from
7 to 12 carbon atoms, or a phenyl or naphthyl group. Examples include methyl, ethyl,
n-propyl,
i-propyl,
n-butyl,
i-butyl,
s-butyl, phenyl, benzyl, phenethyl, 1-phenethyl, 2-phenylpropyl, etc. Moreover, since
the enzymatic reaction of the present invention involves the depicted amino group
and its associated carbon atom, each R¹ and R² group optionally can be substituted
with one or more groups, provided the same are not enzymatically inhibiting groups,
that is, groups which do not significantly affect or compete with the action of the
transaminase when the chiral amine or ketone carrying that group are present in practical
concentrations. This can be readily determined by a simple inhibition assay. Often
when inhibition is detected, it can be minimized by conducting the reaction at lower
concentrations of that reactant. Typical substituents without limitation include
halo such as chloro, fluoro, bromo and iodo, hydroxy, lower alkyl, lower alkoxy, lower
alkylthio, cycloalkyl, carbamoyl, mono- and di-(lower alkyl) substituted carbamoyl,
trifluoromethyl, phenyl, nitro, amino, mono- and di-(lower alkyl) substituted amino,
alkylsulfonyl, arylsulfonyl, alkylcarboxamido, arylcarboxamido, etc.
[0029] Typical groups when R¹ and R² are taken together are 2-methylbutane-1,4-diyl, pentane-1,4-diyl,
hexane-1,4-diyl, hexane-1,5-diyl, and 2-methylpentane-1,5-diyl.
[0030] Typical amines for which the present process is suitable include without limitation
2-aminobutane, 2-amino-1-butanol, 1-amino-1-phenylethane, 1-amino-1-(2-methoxy-5-fluorophenyl)ethane,
1-amino-1-phenylpropane, 1-amino-1-(4-hydroxyphenyl)propane, 1-amino-1-(4-bromophenyl)propane,
1-amino-1-(4-nitrophenyl)propane, 1-phenyl-2-aminopropane, 1-(3-trifluoromethylphenyl)-2-aminopropane,
2-aminopropanol, 1-amino-1-phenylbutane, 1-phenyl-2-aminobutane, 1-(2,5-dimethoxy-4-methylphenyl)-2-aminobutane,
1-phenyl-3-aminobutane, 1-(4-hydroxyphenyl)-3-aminobutane, 1-amino-2-methyl cyclopentane,
1-amino-3-methylcyclopentane, 1-amino-2-methylcyclohexane, and 1-amino-1-(2-naphthyl)ethane.
[0031] In its broadest sense, the process of the first embodiment comprises subjecting
a mixture of chiral amines to the action of an omega-amino acid transaminase which
is enzymatically active (with respect to the depicted amino group of at least one
of said chiral amines) in the presence of an amino acceptor.

in which R¹ and R² are as defined above and, in Formula III, either R³ is R¹ while
R⁴ is R² or R³ is R² while R⁴ is R¹.
[0032] In general, the enzymatic process operates on only one chiral form, or operates on
one chiral form to a far greater extent than the other. For example, with R,S-1-amino-1-phenylethane
(R¹ = phenyl, R² = methyl), only the S-form is converted to the respective nonchiral
ketone, acetophenone, leaving the R-1-amino-1-phenylethane unchanged. Similarly with
R,S-1-amino-1-(4-bromophenyl)ethane (R¹ = 4-bromophenyl, R² = methyl), the S-form
is converted to the non-chiral ketone 4-bromoacetophenone, while R-1-amino-1-4-bromophenyl)ethane
is unchanged. With R,S-1-phenyl-3-aminobutane (R¹ = phenethyl, R² = methyl), the
S-form is readily converted to the nonchiral 1-phenylbutan-3-one whereas the R-form
of 1-phenyl-3-aminobutane is converted to 1-phenylbutan-3-one by a factor of 0.05
or less than that of the S-form.
[0033] In some instances it is possible to assign R¹ and R² configurations to the chiral
amines and identify which is converted to the ketone and which is not. Assignment
of R- and S- designations are made, however, according to the Cahn-Ingold-Prelog method
and depend upon preassigned values for R¹ and R² in the Sequence Rule. Consequently,
a priori assignment of an R- or S- chirality designation to the chiral amine which is acted
upon by the enzyme is not always possible. Hence while assignment of an R- or S- configuration
to the chiral amine of Formula III will depend on the ranking of R³ and R⁴ according
to the Sequence Rule, the configuration of the chiral amine of Formula III will be
identical with one, but only one, of the enantiomers IA and IB. For example and as
noted above, the S-form of 1-amino-1-phenylethane is converted to the nonchiral ketone,
acetophenone, leaving the R-enantiomer unchanged. With R,S-1-amino-1-phenyl-2-hydroxyethane
(phenylglycinol), the enantiomer having the same absolute configuration as that of
1-amino-1-phenylethane is converted but because of the Sequence Rule, this is designated
the R-isomer.
[0034] Since the reaction is an equilibrium, either the forward or reverse reactions can
be favored by the addition of additional starting materials or the removal of reaction
products. When, for example, one desires to enrich the enantiomeric ratio of two chiral
forms of an amine, additional quantities of the amino acceptor can be added (up to
saturation) and/or the ketone formed can be continuously removed from the reaction
mixture. Conversely when one stereoselectively synthesizes one chiral form of an amine,
additional ketone can be added (up to saturation) and/or the amine formed can be removed.
[0035] When the undesired chiral form of the amine is converted to the ketone and the desired
chiral form is not, the latter can be readily isolated by conventional techniques.
Thus a partial separation can be effected by acidification, extraction with a hydrocarbon
such as heptane to remove the ketone, rendering the aqueous phase basic, and re-extraction
with a hydrocarbon such as heptane.
[0036] Often the by-products so isolated are themselves useful commodities. For example,
if the process is practiced so as to enantiomerically enrich a mixture of R-2-aminobutane
and S-2-aminobutane (R¹ = ethyl, R² = methyl) with the R-chiral form, the S-chiral
form will be converted to methyl ethyl ketone, itself a useful organic solvent.
[0037] When, on the other hand, both chiral forms of the amine are desired, the form which
is converted to the ketone can be removed from the reaction mixture (or from the aqueous
phase in a two phase mixture) and independently subjected to the action of an omega-amino
acid transaminase in the presence of a amino donor to generate the same chiral form
as was initially converted to the ketone. For example, starting with a mixture of
R,S-1-amino-1-phenylethane (R¹ = phenyl, R² = methyl), the S-form is converted by
the omega-amino acid transaminase to the respective nonchiral ketone, acetophenone,
leaving the R-1-amino-1-phenylethane unchanged. The R-1-amino-1-phenylethane is readily
isolated from the reaction mixture as described above and the acetophenone by-product
in turn is subjected to the action of the transaminase in the presence of an amino
donor to generate S-1-amino-1-phenylethane in a substantially higher percentage
than is the R-form.
[0038] The second aspect of the foregoing process can be practiced apart from the first.
Hence the stereoselective synthesis of one chiral form of an amine of the formula:

in an amount substantially greater than the other can be achieved by subjecting a
ketone of the formula:
R¹-

-R² II
in which R¹ and R² are as defined above to the action of an omega-amino acid transaminase
in the presence of an amino donor until a substantial amount of one of the chiral
amines is formed. In the example given above, for example acetophenone is subjected
to the action of the transaminase in the presence of an amino donor to generate the
S-1-amino-1-phenylethane exclusive of, or in a substantially higher percentage than,
R-1-amino-1-phenylethane.
[0039] The amino acceptors are ketocarboxylic acids, alkanones, or substances converted
thereto
in situ. Typical of the ketocarboxylic acids are α-keto carboxylic acids such as glyoxalic
acid, pyruvic acid, oxaloacetic acid, and the like, as well as salts thereof. A typical
alkanone is butan-2-one.
[0040] In addition, one can employ other substances which are converted to an amino acceptor
by other enzymes or whole cell processes. Representative of substances converted to
these amino acceptors is fumaric acid (which is rapidly converted to oxaloacetic
acid
in situ), glucose, (which is converted to pyruvate), lactate, maleic acid, etc.
[0041] The amino donors are amines including the nonchiral amino acid glycine and chiral
amino acids having the S-configuration such as L-alanine or L-aspartic acid. Amines,
both chiral and non-chiral, which are not amino acids such as S-2-aminobutane, propyl
amine, benzyl amine, etc. also can be employed.
[0042] Omega-amino acid transaminases useful in the present process are known pyridoxal
phosphate dependent enzymes found in various microorganisms including
Pseudomonas,
Escherichia,
Bacillus,
Saccharomyces,
Hansenula,
Candida,
Streptomyces,
Aspergillus, and
Neurospora. Two omega-amino acid transaminases which are particularly useful in the present
invention, EC 2.6.1.18 and EC 2.6.1.19, have been crystallized and characterized
by Yonaha et al.,
Agric. Biol. Chem,,
47 (10), 2257-2265 (1983).
[0043] Microorganisms having the desired activity can be readily isolated by chemostat
culture, that is, culturing in a constant but restricted chemical environment, with
an amino acceptor and, as the sole nitrogen source, an amine. The amine can be, but
need not be, a chiral amine since in a normal environment omega-amino acid transaminases
metabolize primary amines. Non-chiral amines which have been used successfully to
generate omega-amino acid transaminase include n-octylamine, cyclohexylamine, 1,4-butanediamine,
1,6-hexanediamine, 6-aminohexanoic acid, 4-aminobutyric acid, tyramine, and benzyl
amine. Chiral amines such as 2-aminobutane, α-phenethylamine, and 2-amino-4-phenylbutane
also have been used successfully, as have amino acids such as L-lysine, L-ornithine,
β-alanine, and taurine.
[0044] By such a procedures the culture will be enriched for those microorganisms producing
the desired omega-amino acid transaminases. For example, in one such chemostat conducted
with random soil samples having no particular history of amine exposure was run for
approximately one month. The dominant organisms thereafter were independently identified
by the American Type Culture Collection as
Bacillus megaterium which did not differentiate significantly from and were phenotypically similar to
known strains.
[0045] Organisms so isolated can be grown in a number of ways. Firstly, a standard salts
medium supplemented with phosphate buffer, sodium acetate as a carbon source, 2-ketoglutarate
as an amino acceptor, and a nitrogen-containing compound such as n-propylamine, n-octylamine,
2-aminobutane, 2-aminoheptane, cyclohexylamine, 1,6-hexanediamine, putrescine, 6-aminohexanoic
acid, 4-aminobutyric acid, L-lysine, L-ornithine, β-alanine, α-phenethylamine, 1-phenyl-3-aminobutane,
benzylamine, tyramine, taurine, etc. can be used.
[0046] Alternatively the microorganism can be grown using an amine as the sole carbon source,
thereby limiting growth to those organisms which can catabolize the amine to obtain
carbon.
[0047] Thirdly, the microorganism can be grown using sodium succinate, sodium acetate, or
any other carbon source and an ammonium salt or a protein hydrolysate as the principle
nitrogen source and then adding, either at the outset or during growth, an amine such
as 2-aminobutane, 1-phenyl-3-aminobutane, α-phenethylamine, etc., to induce production
of the desired transaminase activity.
[0048] The actual enzymatic conversion can be effected by conventional culturing techniques
in the presence of the chiral amine, with isolated but non-growing cells, or by bringing
the chiral amines into contact with a soluble omega-amino acid transaminase preparation.
[0049] The omega-amino acid transaminase can be in free form, either as a cell free extract
or a whole cell preparation as noted above, or immobilized on a suitable support or
matrix such as cross-linked dextran or agarose, silica, polyamide, or cellulose. It
also can be encapsulated in polyacrylamide, alginates, fibers, or the like. Methods
for such immobilization are described in the literature (see, for example,
Methods of Enzymology,
44, 1976). The latter embodiment is particularly useful since once the immobilized
enzyme is prepared, one need merely feed the amino acceptor and a mixture of the
chiral amines over the immobilized enzyme in order to effect the desired enrichment,
and then remove the formed ketone in the manner described above.
[0050] Although not necessary, it generally is advantageous to maximize conversion rates
if a source of pyridoxamine, such as pyridoxal phosphate, is included in the reaction
composition.
[0051] Procedures and materials used herein are described below, followed by typical examples.
Procedures and Materials
Enzyme Activity:
[0052] Enzyme activity is expressed herein as units/mg. A unit of enzyme activity is defined
as that which produces 1 micromole of ketone per minute. For uniformity, this is
measured as micromoles of 1-phenylbutan-3-one formed from R,S-1-phenyl-3-aminobutane.
The following standardized assay was utilized to measure the activity of the omega-amino
acid transaminases set forth in the examples which follow.
[0053] A known volume of the enzyme preparation to be tested is incubated at 37°C and pH
7 in a solution having the following composition:
Sodium pyruvate |
100 mM |
R,S-1-Phenyl-3-aminobutane |
30 mM |
Pyridoxal phosphate |
0.5mM |
[0054] A sample is removed and 20% by volume of 12% aqueous trichloroacetic acid are added.
Precipitated protein is removed by centrifugation and the concentration of 1-phenylbutan-3-one
in the supernatant is determined by liquid chromatography on a 100 x 8 mm 4 micron
Novopak phenyl column eluting with 40% isopropanol and 0.09% phosphoric acid in water.
Under these conditions, 1-phenylbutan-3-one elutes at 5.3 minutes.
Purity of Amines:
[0055] The purity of produced amines was determined by gas chromatography on a 6 foot x
2 mm Chrom Q column of 10% SE-30 on a 100/120 mesh support at 210°C with a carrier
gas flow rate of 10 ml/minute.
Determination of Enantiomeric Enrichment:
[0056] The ee of a given product was determined by reacion with (-) α-(trifluoromethylphenyl)methoxyacetyl
chloride {see Gal,
J. Pharm. Sci.,
66, 169 (1977) and Mosher et al.,
J. Org. Chem.,
34, 25430 (1969)} followed by capillary gas chromatography of the derivatized product
on a Chrompack fused silica column.
Standard Salt Medium:
[0057] A suitable salt medium for the microbiological transformations described in the following
examples has the following composition:
MgSO₄ |
1.00g/L |
CaCl₂ |
0.021g/L |
ZnSO₄·7H₂O |
0.20mg/L |
MnSO₄·4H₂O |
0.10mg/L |
H₃BO₃ |
0.02mg/L |
CuSO₄·5H₂O |
0.10mg/L |
CoCl₂·6H₂O |
0.05mg/L |
NiCl₂·6H₂O |
0.01mg/L |
FeSO₄ |
1.50mg/L |
NaMoO₄ |
2.00mg/L |
Fe EDTA |
5.00mg/L |
KH₂PO₄ |
20.00mM |
NaOH |
to pH 7 |
[0058] The composition is not critical but was standardized for all procedures to eliminate
it as a variable.
Microorganisms:
[0059] Cultures either were obtained from the designated depository or were isolated as
described and then independently identified.
Enrichment or Microorganisms Producing omega-Amino Acid Transaminase :
[0060] A chemostat is maintained with 0.5% (w/v) of R,S-2-aminobutane and 10 mM of 2-ketoglutarate
at a dilution rate of 0.03/h in the standard salt medium. The chemostat is inoculated
and run for approximately one month at 37°C and pH 6.8-7.0. Strains which develop
are isolated and grown on minimal agar containing the standard salt medium supplemented
with 10 mM of 2 ketoglutarate and 5 mM of R,S-1-phenyl-3-aminobutane.
Enzyme Recovery:
[0061] Unless otherwise indicated, cells from culture are centrifuged for 10 minutes at
10,000 G, resuspended in 10 mM of phosphate buffer at pH 7 and 0.5 mM of pyridoxal
phosphate, and ruptured by two passes through a chilled French press operating at
15,000 psi. Cell debris is removed by centrifugation for one hour at 10,000 G and
the enzyme-containing supernatant collected.
[0062] The following examples will serve to further typify the nature of this invention
but should not be construed as a limitation on the scope thereof, which is defined
solely by the appended claims.
Example 1
[0063] The following procedure exemplifies the growth of microorganisms producing omega-amino
acid transaminase using an amino donor as the sole source of nitrogen.
[0064] Bacillus megaterium was grown in a 3L shake flask (200 rpm) for 17 hours at 30°C with 1 L of the above
salt solution, 60 mM of sodium acetate, 30 mM of phosphate buffer, 30 mM of disodium
2-ketoglutarate, and 100 mm of n-propylamine as the nitrogen source. When the culture
reached a density of 0.6 g (dry weight)/L, the cells were harvested and the enzyme
isolated as described above. The specific activity of the omega-amino acid transaminase
thus obtained when assayed as above was 0.49 units/mg.
[0065] The
Bacillus megaterium strain used in the foregoing procedure was obtained from soil samples with no particular
history of exposure to amines by inoculating the chemostat previously described and
isolating the dominant organisms (those capable of growing on R,S-1-phenyl-3-aminobutane).
The strain was independently identified by the American Type Culture Collection as
Bacillus megaterium which did not differentiate significantly from the known strain ATCC No. 14581 and
which was phenotypically similar to ATCC 49097
B.
Example 2
[0066] The following procedure exemplifies the growth of microorganisms producing omega-amino
acid transaminase using the amino donor as the sole source of carbon.
[0067] Pseudomonas aeruginosa ATCC 15692 was grown on β-alanine as the sole carbon source as described by Way
et al.,
FEMS Micro. Lett.,
34, 279 (1986) and cell extracts containg the omega-amino acid transaminase then are
obtained as therein described. When assayed as described above, the specific activity
of the omega-amino acid transaminase was found to be 0.040 units/mg.
Example 3
[0068] Pseudomonas putida ATCC 39213 was cultured as described in Example 1 and an enzyme extract then was
obtained as therein described. The specific activity of the omega-amino acid transaminase
was 0.045 units/mg.
Example 4
[0069] The following procedure demonstrates the need for the amino acceptor.
[0070] Enzyme extracts from
P. putida,
B. megaterium, and
P.
aeruginosa obtained as above were assayed as above at pH 9 in 50 mM of Tris/HCl using 30 mM
of R,S-1-phenyl-3-aminobutane, with and without 100 mM of sodium pyruvate. The following
relative rates of conversion were observed.
|
Relative Rate of Conversion |
|
P. putida |
B. megaterium |
P. aeruginosa |
pyruvate |
100 |
100 |
100 |
no pyruvate |
0 |
0 |
0 |
[0071] The transaminase nature of the enzymatic action is apparent from the effect of "suicide
inactivators" known to be specific for transaminases {see, for example, Burnett et
al.,
J. Bio. Chem.,
225, 428-432 (1980)}, the inactivator (0.5 mM) being preincubated with the assay medium
before addition of R,S-1-phenyl-3-aminobutane.
|
Relative Rate of Conversion |
Inactivator |
P. putida |
B. megaterium |
P. aeruginosa |
None |
100 |
100 |
100 |
Gabaculine |
0 |
13 |
0 |
Hydroxylamine |
3 |
10 |
0 |
[0072] The stereoselectivity of the omega-amino acid transaminase can be seen from the
corresponding assay utilizing 15 mM of R-1-phenyl-3-aminobutane (with pyruvate).
|
Relative Rate of Conversion |
|
P. putida |
B. megaterium |
P. aeruginosa |
R,S- |
100 |
100 |
100 |
R- |
3 |
15 |
4 |
Example 5
[0073] The following procedure exemplifies the growth of microorganisms using ammonium as
the sole nitrogen source and then inducing omega-amino acid transaminase production
by the addition of an amine.
[0074] Bacillus megaterium was grown in 1 L cultures in the standard salt medium supplemented with 40 mM of
the indicated carbon source, 5 mM of ammonium chloride, 80 mM of phosphate buffer,
and 2 mM of the amine inducer indicated below. After 30 to 40 hours, the enzyme was
collected and assayed as described above.
|
Specific Activity (units/mg) |
Carbon Source |
Succinate |
Acetate |
Gluconate |
Glucose |
R,S-1-phenyl-1-aminoethane |
0.27 |
0.39 |
n.t. |
n.t. |
R-1-phenyl-1-aminoethane |
0.27 |
0.36 |
n.t. |
n.t. |
R,S-1-phenyl-3-aminobutane |
0.28 |
0.33 |
0.26 |
0.62 |
R-1-phenyl-3-aminobutane |
0.21 |
0.26 |
n.t. |
n.t. |
R,S-2-aminobutane |
0.13 |
0.14 |
n.t |
n.t. |
R-2-aminobutane |
0.06 |
0.13 |
n.t |
n.t. |
tyramine |
n.t. |
0.24 |
n.t |
n.t. |
n.t. = not tested |
Example 6
[0075] The following procedure exemplifies the growth of microorganisms using a protein
rich source and then inducing omega-amino acid transaminase production by the addition
of an amine.
[0076] Bacillus megaterium was grown in 121 L fermenter at pH 7 and 30°C with aeration and agitation in the
above salt medium supplemented with 10 g/L casamino acids. Sodium acetate was added
gradually up to an aggregate concentration of 120 mM. At this point, the cell density
was 3 g (dry weight)/L. 1-Phenyl-3-aminobutane was added up to an aggregate concentration
of 10 mM. After 12 hours, the enzyme was collected and assayed as described above.
The specific activity was 0.49 units/mg.
Example 7
[0077] The following procedure exemplifies the use of a soluble enzyme preparation to effect
enantiomeric enrichment of a racemate of a chiral amine.
[0078] An omega-amino acid transaminase preparation was obtained from
Bacillus megaterium in the manner described in Example 1. Upon assay as described above, it demonstrated
a specific activity of 0.375 units/mg. To a 25 ml. solution of 26.4 mg of this enzyme
preparation, additionally containing 0.4 mM of pyridoxal phosphate and 40 mM of sodium
phosphate, were added 20 mM of R,S-1-amino-1-phenylethane and 100 mM of sodium pyruvate
as the amino acceptor. The solution was incubated for 150 minutes at pH 7 and 30°C
and then rendered alkaline (pH >12) by the addition of 2.5 ml of 2N sodium hydroxide.
The solution was extracted with n-heptane and the extracts evaporated to yield 30.8
mg (49% conversion) of R-1-amino-1-phenylethane having an ee of 96.4%.
Example 8
[0079] The following procedures exemplify the use of a soluble enzyme preparation to effect
enantiomeric enrichment of a racemate of a chiral amine, in each case the recemate
being subatituted for for R,S-1-amino-1-phenylethane in the procedure of Example
7:
Starting Material
[0080]
(a) R,S-1 phenyl-3-aminobutane
(b) R,S-1-amino-1-(4-bromophenyl)ethane
(c) R,S-1-phenyl-2-aminopropane
(d) R,S-1-amino-1-phenylethane
(e) R,S-4-(4-methoxyphenyl)-2-aminobutane
(f) R,S-5-(3-pyridyl)-2-aminopentane
Product |
|
ee |
% Conversion |
(a) R-1-phenyl-3-aminobutane |
98.4 |
60 |
(b) R-1-amino-1-(4-bromophenyl)ethane |
97.6 |
49 |
(c) R-1-phenyl-2-aminopropane |
98.6 |
49 |
(d) R-1-amino-1-phenylethane |
99 |
52 |
(e) R-4-(4-methoxyphenyl)-2-aminobutane |
99 |
58 |
(f) R-5-(3-pyridyl)-2-aminopentane |
99 |
49 |
Example 9
[0081] The following procedure exemplifies the use of non-growing cells to effect enantiomeric
enrichment of a racemate of a chiral amine.
[0082] The cells from three 1 L cultures of Bacillus megaterium grown for 33 hours in the
manner described in Example 1 on 6 mM of R,S-1-phenyl-3-aminobutane as the sole nitrogen
source were harvested by centrifugation and washed by resus pension in 250 ml of
10 mM phosphate buffer (pH 6.8) and centrifugation.
[0083] The cell pellet was resuspended in 0.6 L of 10 mM phosphate buffer (pH 6.8) containing
10 mM of R,S-1-phenyl-3-aminobutane and 50 mM of oxaloacetic acid as the amino acceptor.
After incubation on an orbital incubator at 30°C for 4 hours, the solution was rendered
alkaline and extracted with heptane as described in Example 7. R-1-Phenyl-3-aminobutane
thus was obtained in 97.9% optical purity, corresponding to an ee of 95.8.
Example 10
[0084] The following procedure exemplifies the use of growing cells to effect enantiomeric
enrichment of a racemate of a chiral amine and the use of an amino acceptor precursor.
[0085] A 6 L innoculum of
Bacillus megaterium, prepared substantially as described in Example 1 but using 10 mM of R,S-1-phenyl-3-aminobutane
as the sole nitrogen source, was cultured in a 120 L volume of the above salt medium
supplemented with 30 mM of fumarate as the amino acceptor precursor. Twenty-two
hours after inoculation, an additional 30 mM of fumarate was added and 6 hours later
the culture was harvested by removing the cells through ultrafiltration using a Romicon
PM100 membrane. The solution was rendered alkaline and extracted with heptane as described
in Example 7. R-1-Phenyl-3-aminobutane thus was obtained in 99.5% purity with an
ee of 96.4%.
Example 11
[0086] The following procedure exemplifies the relative rates of conversion, determined
directly or calculated from kinetic data, of different chiral amines by soluble enzymatic
preparations utilizing the assay described above but substituting the indicated chiral
amine.
|
|
Relative Rate of Conversion |
Amine (R,S) |
Conc. (mM) |
R-enantiomer |
S-enantiomer |
1-phenyl-1-aminoethane |
10 |
0 |
100 |
1-phenyl-3-aminobutane |
30 |
5 |
100 |
1-(4-bromophenyl)-1-aminoethane |
30 |
0 |
100 |
1-(α-naphthyl) 1-aminoethane |
10 |
0 |
100 |
phenylglycinol |
10 |
100 |
0 |
2-aminooctane |
5 |
0 |
100 |
5-(3-pyridyl)-2-aminopentane |
5 |
0 |
100 |
1-(4-nitrophenyl)-2-aminopropane |
5 |
0 |
100 |
3-phenyl-2-aminopropane |
15 |
7 |
100 |
1-phenyl-1-aminopropane |
20 |
11 |
100 |
3-phenyl-2-aminopropane |
10 |
100 |
0 |
Example 12
[0087] The following procedure exemplifies the relative rates of conversion with 1-phenyl-3-aminobutane
employing differ ent amino acceptors in place of pyruvate in the assay described
above.
Acceptor |
Conc. (mM) |
Relative Rate of Conversion |
pyruvate |
20 |
100 |
oxaloacetate |
20 |
100 |
heptaldehyde |
25 |
80 |
glyoxalate |
20 |
50 |
2-ketobutyrate |
25 |
21 |
butan-2-one |
20 |
20 |
acetaldehyde |
20 |
50 |
propionaldehyde |
20 |
100 |
butyraldehyde |
20 |
90 |
benzaldehyde |
25 |
17 |
2-pentanone |
25 |
33 |
cyclopentanone |
25 |
12 |
cyclohexanone |
25 |
23 |
hydroxypyruvate |
25 |
18 |
[0088] Also found to be effective as amino acceptors although considerably less so (relative
rates = <10) is acetophenone.
Example 13
[0089] The following procedure exemplifies enantiomeric enrichment using a soluble enzyme
preparation with continuous extraction of the enriched product.
[0090] A soluble enzyme preparation was obtained from
Bacillus megatarium in the manner described in Example 1. Upon assay as described above, it demonstrated
a specific activity of 0.70 units/mg. An aqueous phase was prepared containing 450
mg of this extract, 0.12 M sodium pyruvate, 0.2 M R,S-1-phenyl-3-aminobutane, 1mM
pyridoxal phosphate, and 0.5 M phosphate (pH 7.5). Five hundred milliliters of n-heptane
were added and the two phase mixture was stirred at 22°C for seven hours. The pH was
then adjusted to 4.5 by the addition of hydrochloric acid and the aqueous layer was
separated from the organic layer. The aqueous layer was rendered alkaline by the
addition of sodium hydroxide and extracted with heptane. Upon removal of the heptane,
the residue was analyzed as containing 96% R-1-phenyl-3-aminobutane.
Example 14
[0091] The following procedure typifies the synthesis of a chiral amine.
[0092] A soluble enzyme preparation was obtained from
Bacillus megatarium in the manner described in Example 1. Upon assay as described above, it demonstrated
a specific activity of 0.58 units/mg. To a 200 ml aqueous solution of 350 mg of this
preparation, 0.4 mM of pyridoxal phosphate, and 40 mM of sodium phosphate, are added
4.2 mM of 1-phenylbutan-3-one and 100 ml of 2-aminobutane as the amine donor. The
mixture was incubated at pH 7 and 30°C for 4 hours, at which point R-1-phenyl-3-aminobutane
was present in the reaction mixture at a concentration of 3.35 mM, corresponding to
80% conversion. The product was isolated by the addition of 40 ml of 10 N sodium
hydroxide and extraction of the alkaline aqueous solution with 250 ml of n-heptane.
Upon evaporation of the heptane extracts, there were obtained 100.5 g of product which
was analyzed by derivation as previously described and found to contain 96.4% of S-1-phenyl-3-aminobutane.
[0093] Similarly, S-1-phenyl-2-aminopropane was prepared from 1-phenylpropan-2-one at an
ee of 96.4 and a yield of 94.8%. S-1-amino-1-phenylethane was prepared from acetophenone
at an ee of 100 and a yield of 44%.
Example 15
[0094] This procedure exemplifies the enzymatic separation and isolation of each of the
R- and S-enantiomers.
[0095] The procedure for obtaining the R-enantiomer of R,S-1-amino-1-phenylethane described
in Example 7 is followed through the incubation. Prior to rendering the incubation
solution alkaline, however, it is extracted with n-heptane and the extracts are retained.
The aqueous phase then is processed according to Example 7 to isolate R-1-amino-1-phenylethane
as described therein.
[0096] Acetophenone is recovered from the retained heptane extracts by evaporation. By
following substantially the same procedure as set forth in Example 14 but employing
2.3 mM of acetophenone in place of 1-phenylbutan-3-one, 56 mg of S-1-amino-1-phenylethane
(100%) were obtained.
Example 16
[0097] This procedure exemplifies the use of immobilized enzyme.
Immobilization:
[0098] A 47 mm diameter ACTIDISK (FMC Corp.) support matrix (0.4 g) was loaded into a housing
(Millipore Sweenex) fitted with inlet and outlet tubing, a peristaltic pump, and reservoir.
The matrix was washed sequentially, at ambient temperatures and a rate of 3 ml/min.,
with (1) 200 ml of 50 mM of phosphate buffer (pH 7) containing 0.5 mM pyridoxal phosphate
over a period of 20 min., (2) 11 ml of a 4.6 mg/ml solution of enzyme obtained in
the manner of Example 1 for 120 min., (3) 150 ml of 0.3 M sodium chloride in 50 mM
of phosphate buffer (pH 7) containing 0.5 mM pyridoxal phosphate for 30 minutes, and
(4) 200 ml of 50 mM of phosphate buffer (pH 7) containing 0.5 mM pyridoxal phosphate
over a period of 20 min.
Enrichment:
[0099] A 140 ml. solution of 10 mM R,S-1-phenyl-3-aminobutane, 100 M of sodium pyruvate,
0.1 mM of pyridoxal phosphate, and 25 mM of potassium phosphate (pH 7) was circulated
through the above matrix at ambient temperatures and a rate of 5 ml/min. After two
hours, the circulating liquid was removed from the apparatus. The concentration of
1-phenylbutan-3-one formed was 5.2 mM while that of R-1-phenyl-3-aminobutane was
4.8 mM. The pH was adjusted to 12.5 and R-1-phenyl-3-aminobutane was isolated quantitatively
by extraction with heptane. After removal of the heptane by evaporation, the product
was analyzed as 92.8% R-1-phenyl-3-aminobutane.
1. A process for the enantiomeric enrichment of a mixture of two enantiomeric chiral
amines of the formula:

in which each of R¹ and R² is an alkyl or aryl group which is unsubstituted or substituted
with an enzymatically non-inhibiting group and R¹ is different from R² in structure
or chirality,
which comprises bringing said mixture of chiral amines, in an aqueous medium and in
the presence of an amino acceptor, into contact with an omega-amino acid transaminase
which is enzymatically active with respect to the depicted amino group of one of said
chiral amines, at least until a substantial amount of one of said chiral amines is
converted to a ketone of the formula:
R¹-

-R²
in which R¹ and R² are as defined for said amine.
2. The process of claim 1 in which said contact is maintained at least until the
enantiomeric excess of the chiral amine which is not converted to said ketone is at
least about 90% relative to the other chiral amine.
3. The process according to claim 1 wherein the chiral amine which is not converted
to said ketone is recovered from the reaction mixture.
4. The process according to claim 1 wherein a substantial quantity of said ketone
is recovered from the aqueous media.
5. The process according to claim 4 wherein the ketone recovered from the aqueous
media is independently brought into contact with an omega-amino acid transaminase
in the presence of an amine donor at least until the same chiral form as was initially
converted to said ketone is formed in an amount substantially greater than the other
chiral form is formed.
6. The process according to claim 1 wherein the amino acceptor is an α-keto carboxylic
acid, an aliphatic or cycloaliphatic ketone, an aliphatic or cycloaliphatic aldehyde,
or a substance which is biochemically converted to an α-keto carboxylic acid in situ in the reaction medium.
7. The process according to claim 6 wherein the amino acceptor is glyoxalic acid,
pyruvic acid, oxaloacetic acid, a salt thereof, or heptaldehyde.
8. The process according to claim 1 wherein each of R¹ and R² independently is a straight
or branched alkyl group of from 1 to 6 carbon atoms, a straight or branched phenylalkyl
group of from 7 to 12 carbon atoms, or a phenyl or naphthyl group, each of said groups
being unsubstituted or substituted with an enzymatically non-inhibiting group.
9. The process according to claim 8 wherein each of R¹ and R² independently is methyl,
ethyl, n-propyl, isopropyl, n-butyl, i-butyl, s-butyl, phenyl, benzyl, or phenethyl.
10. The process according to claim 1 wherein said mixture of chiral amines and amino
acceptor are brought into contact with whole cells of a microorganism which produces
omega-amino acid transaminase.
11. The process according to claim 1 wherein said mixture of chiral amines and amino
acceptor are brought into contact with a cell-free aqueous preparation of said omega-amino
acid transaminase.
12. The process according to claim 1 wherein said mixture of chiral amines and amino
acceptor are brought into contact with said omega-amino acid transaminase immobilized
on a support.
13. The process for the stereoselective synthesis of one chiral form of an amine of
the formula:

in an amount substantially greater than the other, in which each of R¹ and R² is
an alkyl or aryl group which is unsubstituted or substituted with an enzymatically
non-inhibiting group and R¹ is different from R² in structure or chirality, which
comprises bringing a ketone of the formula:
R¹-

-R²
in which R¹ and R² are as defined for said amine into contact with an omega-amino
acid transaminase in the presence of an amino donor at least until a substantial amount
of one of said chiral amines is formed.
14. The process of claim 13 in which the amino donor is 2-aminobutane, glycine, alanine,
or aspartic acid.
15. The process according to claim 13 wherein each of R¹ and R² independently is a
straight or branched alkyl group of from 1 to 6 carbon atoms, a straight or branched
phenylalkyl group of from 7 to 12 carbon atoms, or a phenyl or naphthyl group, each
of said groups being unsubstituted or substituted with an enzymatically non-inhibiting
group.
16. The process according to claim 15 wherein each of R¹ and R² independently is methyl,
ethyl, n-propyl, isopropyl, n-butyl, i-butyl, s-butyl, phenyl, benzyl, or phenethyl.
17. The process according to claim 13 wherein said ketone and amino donor are brought
into contact with whole cells of a microorganism which produces omega-amino acid transaminase.
18. The process according to claim 13 wherein said ketone and amino donor are brought
into contact with a cell-free aqueous preparation of said omega-amino acid transaminase.
19. The process according to claim 13 wherein said ketone and amino donor are brought
into contact with said omega-amino acid transaminase immobilized on a support.
20. The process according to claim 13 wherein a large molar excess of said amino donor
is employed.